CN111418558B - Construction method of ablation metabolic urinary calculus animal model - Google Patents
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- 208000009911 Urinary Calculi Diseases 0.000 title claims abstract description 22
- 238000010171 animal model Methods 0.000 title claims abstract description 18
- 206010007027 Calculus urinary Diseases 0.000 title claims abstract description 16
- 230000002503 metabolic effect Effects 0.000 title claims abstract description 15
- 238000010276 construction Methods 0.000 title claims abstract description 10
- 238000002679 ablation Methods 0.000 title abstract description 17
- QXDMQSPYEZFLGF-UHFFFAOYSA-L calcium oxalate Chemical compound [Ca+2].[O-]C(=O)C([O-])=O QXDMQSPYEZFLGF-UHFFFAOYSA-L 0.000 claims abstract description 26
- 239000013078 crystal Substances 0.000 claims abstract description 21
- 229960002635 potassium citrate Drugs 0.000 claims abstract description 21
- 239000001508 potassium citrate Substances 0.000 claims abstract description 21
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 claims abstract description 21
- 235000011082 potassium citrates Nutrition 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 16
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- 241000209094 Oryza Species 0.000 claims description 29
- 235000009566 rice Nutrition 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 28
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 20
- 241000238631 Hexapoda Species 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
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- 241000209140 Triticum Species 0.000 claims description 4
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 230000001788 irregular Effects 0.000 claims description 3
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
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- VBUNOIXRZNJNAD-UHFFFAOYSA-N ponazuril Chemical compound CC1=CC(N2C(N(C)C(=O)NC2=O)=O)=CC=C1OC1=CC=C(S(=O)(=O)C(F)(F)F)C=C1 VBUNOIXRZNJNAD-UHFFFAOYSA-N 0.000 claims 1
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- 150000001450 anions Chemical class 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
A construction method of an animal model for ablating metabolic urinary calculi belongs to the field of biomedicine; the method is characterized in that potassium citrate solution with the concentration of 6.7-12.8 mM and the volume of 5-10 mu L is injected into an animal model for constructing the urinary calculus, and calcium oxalate crystals formed in a mah-jong tube of the animal model are gradually disintegrated after the potassium citrate solution is injected for 12 hours until the calcium oxalate crystals completely disappear. The invention is to control the forming process of calcium oxalate crystals, and can regulate the processes of gradually disintegrating and melting the formed calcium oxalate until the calcium oxalate is eliminated. The animal model construction method has the advantages of simple operation, short implementation period, easily obtained materials, reversible and controllable process and the like, and is suitable for primary or metabolic urinary calculus formation and ablation process thereof, pathological analysis and drug test and screening.
Description
Technical Field
The invention belongs to the field of biomedical research, and particularly relates to a construction method of an ablation metabolic urinary calculus animal model.
Background
The primary or metabolic urinary calculus mainly comprises urinary calculus and kidney calculus, which are mostly generated in people with little water intake, the calculus incidence rate is up to 11%, and in the existing urinary calculus and kidney calculus, calcium oxalate alone or together with other calcium salts forms the main component of urinary calculus. The human being has particularity as a treatment object, deeply understands and researches the formation, prevention and ablation process of the metabolic urinary calculus and needs to rely on a proper animal model to carry out corresponding scientific research. According to the symptom characteristics of calculus in different tissues and organs, different animal models have been constructed using animals such as mice, rabbits, pigs, etc. In order to better understand and understand primary or metabolic urinary calculi, especially the ablation process and physiological mechanism of calculi, it is objectively necessary to construct diversified animal models.
Drosophila became the internationally accepted classical insect model for studying animal diseases and life laws, and provides important reference data for disease occurrence mechanism, treatment scheme and drug screening. The locusta chinensis is a common agricultural pest in a paddy field, and takes stems and leaves of the paddy rice, wherein dozens of hollow tubules with blind ends grow at the junction of intestines and hindgut, and the tubules are dissociated in haemolymph and automatically crawl. The developmental theory and the evolutionary theory believe that the insect Mariothis marxiana and the mammalian kidney are homologous organs, and the genetic background, the physiological structure and the function of the insect Mariothis marxiana are extremely similar, so the Mariothis marxiana is regarded as a detoxifying and urination organ of the insect, small molecule metabolites generated by various tissues in vivo can be absorbed from the haemolymph into the tube to form the prourine, and are combined with midgut metabolites to form the excrement to be discharged out of the body.
In the previous research, the inventor proves that calcium oxalate is formed in the Ma's tube of the Chinese rice locust stressed by copper chloride, and after a certain amount of potassium citrate is injected into the haemolymph of the Chinese rice locust which generates calcium oxalate crystals, the calcium oxalate is gradually ablated. The construction of the insect urinary system stone model based on the physiological process is scientific. The model is applied to develop the ablation process and mechanism of urinary calculus, and provides a test model and a practical carrier for preventing and treating primary or metabolic urinary calculus and screening urinary calculus ablation drugs.
Disclosure of Invention
The invention aims to provide a construction method of an animal model for ablating metabolic urinary calculi, which is a method for ablating calcium oxalate crystals generated in a Ma's tube of a Chinese rice locust by potassium citrate with a certain concentration according to the physiological metabolism rule of the Chinese rice locust on metal ions and application thereof.
A construction method of an animal model for ablating metabolic urinary calculi comprises the following contents:
1. using wild captured adult Chinese rice locust, and adaptively feeding with fresh wheat seedling in a climatic incubator for 2-3 days;
2. the environmental conditions for raising the Chinese rice locust are as follows: the illumination and dark time is 14 h to 10 h, the temperature in illumination is 25-28 +/-2 ℃, the temperature in dark is 21-26 +/-2 ℃, and the humidity is 65-85%;
3. a micro-injection needle penetrates into an internode membrane from the 3 rd and 4 th abdominal joints of the insect abdomen along the direction opposite to the abdomen extension direction and slowly injects a copper chloride solution;
4. the copper chloride solution injected into abdominal cavity of Oxya chinensis (Miq.) Kuntze is prepared from insect normal saline, and is sterilized by steam at 120 deg.C for 20 min;
5. the formula of the insect normal saline comprises: NaCl: 0.138M, Na2HPO4：0.0073 M, KH2PO4：0.00147 M，KCl：0.0027 M， pH=7.4；
6. Sterilizing with microinjector and insect at 75%, and injecting copper chloride solution into abdominal cavity of Aconitum chinensis (Ornithogalum chinense (L.) Kuntze) at concentration of 6.0-10.5 mM and injection dosage of 4-8 μ L;
7. feeding for 1 day after injecting copper chloride solution, and performing secondary injection of medicinal copper chloride solution by the same method;
8. the parameters of the insect breeding environment after injection are the same as those in the step 2;
9. after the rice is raised for 24 to 48 hours, irregular calcium oxalate crystals are formed in the Ma-tube of the Chinese rice locust, and the crystals show blue fluorescence under ultraviolet and visible light and are cuboid or irregular;
10. the potassium citrate solution is prepared by insect normal saline with the concentration of 12.8 mM, and then is sterilized by steam at 120 ℃ for 20 min;
11. injecting 6.7-12.8 mM potassium citrate solution into Chinese rice locust producing calcium oxalate crystals, wherein the injection volume is 5-10 μ L, and feeding in a climatic incubator;
12. after the Chinese rice locust injected with the potassium citrate solution is raised for 12 hours, dissection and the Marsdenia tenacissima is observed through a microscope, so that calcium oxalate crystals begin to melt and gradually decrease with the time.
Compared with other existing animal models, the invention has the advantages that the adopted Chinese rice locust is a rice field insect, the copper chloride and the potassium citrate are conventional reagents, and the material cost is low; the model construction operation is simple, the formation and ablation repeatability of calcium oxalate crystals are good, the period is short, and the related indexes are convenient to quantify; the individual of the Chinese rice locust is more than 20 times larger than that of the fruit fly, the number of the Martes tube is large, the crystal is convenient to dissect, observe and separate, and the ablation process of the metabolic urinary calculus-calcium oxalate crystal is convenient to monitor in real time.
The invention regulates and controls the ablation speed of calcium oxalate crystals by controlling the dosage and the action time of the potassium citrate solution. Can be applied to the physiological function of potassium citrate in the urinary system and the change mechanism of the related anion/cation concentration in the ablation process of calcium oxalate to carry out systematic research. The invention constructs a good animal model for carrying out ablation and disease treatment of human metabolic urinary calculi, drug screening and pharmacokinetic research.
Drawings
FIG. 1 shows that calcium oxalate crystals in Ma-tube of Chinese rice locust are gradually ablated under the action of potassium citrate.
Detailed Description
Example 1:
1. collecting adult Chinese rice locust in a field rice field, putting the adult Chinese rice locust into a creel, adaptively feeding the adult Chinese rice locust in a climatic incubator for 2-3 days, and feeding the adult Chinese rice locust with fresh wheat seedlings in the period, wherein the preferable feeding conditions are as follows: the light and dark are 13:11 hours, the temperature corresponding to the photoperiod is 25 +/-2 ℃ and 21 +/-2 ℃, and the humidity is 70-80 percent;
2. the formula of the insect normal saline comprises: NaCl: 0.138M, Na2HPO4：0.0073 M, KH2PO4：0.00147 M，KCl：0.0027 M， pH=7.4；
3. Preparing 8.5 mM copper chloride solution and 10.0 mM potassium citrate solution with the insect normal saline, and sterilizing for later use by steam sterilization at 120 ℃ for 20 min;
4. rinsing body surface and microinjector with 75% alcohol for sterilization, picking adaptively raised Chinese rice locust, penetrating internode membrane from the 3 rd and 4 th abdominal nodes of Chinese rice locust with microinjector, and slowly injecting 8 μ L of copper chloride solution with concentration of 8.0 mM;
5. 1-2 days later, insects are dissected, and transparent calcium oxalate crystals with regular shapes are generated in Marsdenia tubes under a microscope;
6. preparing 12.8 mM potassium citrate solution with insect normal saline, and sterilizing with 120 deg.C steam for 20 min;
7. injecting 8 μ L potassium citrate solution with concentration of 7.0 mM into Chinese rice locust with calcium oxalate nodule generation, placing in a climatic incubator, and feeding with fresh wheat seedling;
8. after 12 hours of the Chinese rice locust injected with the potassium citrate solution, the decomposition of calcium oxalate crystals in the Martensis tube can be observed through a microscope; the disintegration or ablation process of calcium oxalate can be regulated by adjusting the dosage of potassium citrate and the feeding amount.
Example 2: in this example, the concentration of the cupric chloride solution injected into the abdominal cavity of a chinese rice locust was 7.0 mM, the injection amount was 4 μ L, the concentration of the potassium citrate solution injected after 1 day of the raising was 7.0 mM, the injection amount was 5 μ L, and the same effects as in example 1 were obtained except that the requirements were the same as in example 1.
Example 3: in this example, the concentration of the cupric chloride solution injected into the abdominal cavity of the locusta chinese was 6.0 mM, the injection amount was 7 μ L, the concentration of the potassium citrate solution injected after 1 day of the raising was 6.0 mM, the injection amount was 6 μ L, the other operational requirements were the same as those of example 1, and the calcium oxalate crystal ablation rate was slower and the period could be extended by 1 to 2 days as compared with example 1.
Example 4: in this example, the concentration of the cupric chloride solution injected into the abdominal cavity of the locusta chinese was 10.0 mM, the injection amount was 4 μ L, the concentration of the potassium citrate solution injected after 1 day of raising was 7.0 mM, the injection amount was 8 μ L, other operation requirements were the same as those of example 1, the calcium oxalate crystal ablation rate was accelerated, and the complete ablation was carried out 1 to 2 days earlier than that of example 1.
Claims (3)
1. A construction method of an animal model for ablating metabolic urinary calculus comprises injecting potassium citrate solution with concentration of 6.7-12.8 mM and volume of 5-10 μ L into the animal model for constructing urinary calculus, and gradually disintegrating calcium oxalate crystals formed in a marquis tube of the animal model after injecting the potassium citrate solution for 12 hours until the calcium oxalate crystals completely disappear; the method is characterized by comprising the following steps of:
(1) adaptively feeding adult Chinese rice locust in a climatic incubator for 2-3 days by using fresh wheat seedlings; the breeding conditions are as follows: the period of light and dark is 13:11 hours, the temperature in the corresponding light is 25-28 +/-2 ℃, the temperature in the dark is 21-26 +/-2 ℃, and the humidity is 65-85%;
(2) spraying 75% alcohol on the body surface of Chinese rice locust for disinfection; sterilizing the syringe with 75% alcohol;
(3) injecting 4-8 μ L of copper chloride solution with concentration of 6.0-10.5 mM into internode membrane of 3 rd and 4 th abdominal nodes of Oxya chinensis (Oryza Glutinosa) by syringe;
(4) feeding Chinese rice locust injected with copper chloride solution in a climatic incubator for 1 day, injecting copper chloride solution 4-6 μ L, and feeding for 1-2 days; irregular calcium oxalate crystals are formed in the Mactra tube of the Chinese rice locust.
2. The method of claim 1, wherein the cupric chloride solution is prepared with normal saline insect solution at a concentration of 10.5 mM and steam sterilized at 120 deg.C for 20 min.
3. The method for constructing an animal model for ablating metabolic urinary calculi according to claim 2, wherein the formulation of the insect physiological saline is as follows: NaCl: 0.138M, KCl: 0.0027M, Na2HPO4：0.0073 M，KH2PO4：0.00147 M，pH=7.4。
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